On the results reported below the plant extracts used according to the present invention are identified by their respective original coded name, with the following correspondence:                L-18: Artocarpus heterophyllus seed extract,        L-19: Cyathea cumingii leaf extract (polar solvent extraction),        L-20: Secale cereale seed extract (polar solvents extraction, in particular water, followed by an enzymatic hydrolysis),        L-21: Secretion of Thalassiosira pseudonana (obtained from a culture in sea water),        L-22: Soy seed pericarp extract (aqueous suspension followed by an enzymatic hydrolysis),        L-23: Buddleja axillaris leaf extract (extraction/concentration, then lyophilisation/sterilisation).        
This report presents data on the effect of various L-18-23 on melanosome transfer in normal human epidermal melanocytes to fully matched epidermal keratinocytes.
The optimal doses have been standardized for each plant extract active separately on epidermal melanocytes and keratinocytes. The effect of all L-18-23 on a) melanin synthesis and b) tyrosinase activity using melanocytes cultured alone and cultured together with matched keratinocytes (i.e., EM-KC co-cultures) has been assessed. The effects of the plant extracts actives on melanosome transfer in EM: KC co-culture using gp00 immunolabelling has been evaluated.
Both positive melanocyte modulator (IBMX) and a melanosome transfer inhibitor (Niacinamide) were used as control for this study.
In general, L-18, 19, 20, 21, 23 downregulated melanin synthesis, dopa-oxidase activity of tyrosinase, melanosome transfer, and Myo-X expression. In fact, L-18 and L-23 were known as depigmentating agents but their action on these parameters was not shown so far. By contrast, L-22 was found to upregulate said parameters.
Materials & Methods
Stimulation of Melanocytes and Melanoma Cells with Plant Extracts of the Invention.
Cell Culture:
In the report below, the starting material used                for L-18, is a powder (put in aqueous mother solution, then adjusted to the indicated concentration),        for L-19, is a solution in a water/glycerol mixture at dry extract 30%,        for L-20, is an aqueous solution at dry extract 5.6%,        for L-21, is an aqueous solution at dry extract 6%,        for L-22, is an aqueous solution at dry extract 15%,        for L-23, is a powder (put in aqueous mother solution, then adjusted to the indicated, concentration),        
Assessment of doses: Fully-matched epidermal melanocytes EM and epidermal keratinocytes (EK) were seeded into 6-well plates in serum-supplemented full MEM melanocyte and K-SFM keratinocyte medium for 24 h. The cells were switched to serum free medium (so-called starved) supplemented with L-18 (0.001-0.005%), L-19 (0.5-1.0%), L-20 (0.1-0.5%), L-21 (10 μg/ml), L-22 (0.1-1.0%) and L-23 (0.01-0.05 μg/ml) for 12 and 72 h. Controls included IBMX (100 μM) and niacinamide (10 μM). Cytotoxicity was assessed by cell death and cytopathologic change in morphology.
Approximately 1×104 EM and 2×103 FM55 melanoma cells were seeded into each well of a Lab-tek® 8-well chamber slide and allowed to attach for 24 h. Cells were then washed 3-times with sterile PBS and supplemented with 350 μl of either fresh serum-free RPMI medium (melanoma cells) or starved serum-free and BPE-free melanocyte medium (EM) and incubated at 37° C. and 5% CO2 for 24 h. Cells were then washed with sterile PBS 3-times and incubated with IBMX 1×10−4 M at 37° C. and 5% CO2 for 12, 24, and 72 h. Cells were then gently washed 3-times with sterile PBS and fixed in ice-cold methanol for 10 minutes at −20° C. Slides were stored at −20° C. until immunocytochemistry was performed.
Melanin Assay: 500 μg/ml of synthetic melanin (Sigma, UK) was prepared in 1 M sodium hydroxide (NaOH) (BOH Ltd, UK) and dissolved in a sonicating water bath for 20 minutes. From this stock solution, various melanin standards were prepared in 1 M NaOH from 50 μg/ml to 1 μg/ml. The melanin standards were pipetted into a 96 well plate to produce a calibration curve for the assessment of melanin content in the test samples. 400 μl of 1 M NaOH was added to each cell pellet and dissolved on a heat block (100° C.) for 15 minutes. The pellets were vortexed vigorously and the solubilized pellet was pipetted into the same 96-well plate. The optical densities of the sample were read at 495 nm on a DYNEX REVELATION 4.02 program. Melanin content of each test sample was read from the calibration curve.
DOPA oxidase detection using non-denaturing SDS-PAGE for assessment of tyrosinase activity: Approximately 5×105 of EMc, and FM55 melanoma cells were seeded into three T75 flasks and were incubated at 37° C. and 5% CO2 overnight. The cells were prepared for SDS-PAGE and transblotted onto PVDF membranes. 70 μg of un-reduced protein extract without boiling was pipetted into the appropriate wells of 8% SDS-PAGE gels. The PVDF membrane containing the separated proteins was washed once in 1× PBS and then incubated at RT in 5 mM L-DOPA in 0.1 M sodium phosphate buffer for 3 hours with three changes of the L-DOPA. The L-DOPA reaction was stopped by washing the membrane in distilled water and the membrane was scanned.
Western blot analysis: EMc in a confluent T225 flask were trypsinised and seeded into T25 flask with full medium and allowed to attach overnight. 24 h before treatment the medium was replaced with starved medium for 24 h. Cells were washed 3-times with sterile PBS, and incubated in starved medium alone, or L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml) for 12 and 72 h. Controls included IBMX (100 μM) and niacinamide (10 μM).
40 μg of total protein from each cell extract was electrophoresed in reducing SDS-8%-PAGE and blotted on PVDF membranes (Millipore Corporation, Bedford, Mass.). The membranes were blocked with 5% milk PBS/0.075% Tween 20 for 2 h at room temperature and were then probed with primary antibodies for overnight at 4° C. The molecular weight ladder (Magik Marker, Invitrogen) was incubated in 5% milk PBS/0.075% Tween 20 and the membrane strips were incubated with either 1 ml of 5% milk PBS/0.075% Tween 20 (negative control), or 1 ml of rabbit anti-Myo-X polyclonal antibody (1:200) in 5% milk PBS/0.075% Tween 20), 1 ml of rabbit anti-fascin (1:500) polyclonal antibody in 5% milk PBS/0.075% Tween 20) and 1 ml of goat anti-actin (1:1000) polyclonal antibody in 5% milk PBS/0.075% Tween 20) on a rocking platform overnight at 4° C.
After extensive washes the blots were incubated with horseradish peroxidise conjugated secondary antibodies for 2 h. The molecular weight ladder was then incubated with 1 ml of HRP-goat anti-human IgG (H+L) (Zymed, USA) (1:500) diluted in 5% milk PBS/0.075% Tween 20 and membrane strips were incubated in 1 ml of anti-rabbit IgG, horseradish peroxidase linked whole antibody (HRP) secondary antibody (Amersham Biosciences, UK) (1:700 diluted in 5% milk PBS/0.075% Tween 20) on a rocking platform for 2 hours at room temperature. The washing procedure was repeated and the membrane strips were incubated in LumiGLO® Reagent and Peroxide (BioLab Ltd, UK) for 2 minutes at room temperature. The chemiluminescent signal was detected by exposing the blot strips to Kodak XRA X-ray film (Kodak, UK) at various exposure times, followed by development in developing solution (Kodak, UK) until bands appeared, rinsed in tap water, fixed in the fixer (Kodak, UK) until the film turned blue, then rinsed with tap water and allowed to dry. The membrane was then labelled and scanned densitometric analysis a software (Image Master Total lab version 1.11).
Immunofluorescence staining: For EM:KC co-culture studies fully matched melanocytes (p4) and keratinocytes (p2) were seeded onto 8-well Lab-Tek® chamber slides at a cell density of 1×104 cells/well and in a ratio of 1 melanocyte to 10 keratinocytes. Co-cultures were maintained overnight (16 h) in a mixture of full K-SFM and MEM (co-culture medium) to allow cell attachment, followed by medium replenishment for a further 24 h. Cells were then fixed in ice-cold methanol for 10 min at −20° C., washed in PBS and then blocked with 10% donkey serum for the detection of protein expression in filopodia.
For double labelling experiments the first primary antibody, Myo-X (1:100) (Santa Cruz, Calif., USA) was applied overnight at 4° C., followed by incubation with FITC-conjugated secondary antibody (1:100) for 1 h at room temperature. The second primary antibody, either cytokeratin (1:100) (Abeam, Cambridge, UK) or β-Actin (1:100) (Santa Cruz, USA) or Fascin (1:100) (Santa Cruz, USA) or NKi/beteb (1:30) (Monosan) was applied for 1 h at room temperature followed by a TRITC-conjugated secondary antibody (1:100) (Jackson Immunoresearch Laboratories, Inc., West Grove, USA). DAPI (Vector Laboratories, Burlingame, Calif.) was used to stain nuclei. Images were captured with a cooled Hamamatsu digital camera using a 100× objective and post-processed using Paint Shop Pro (Jasc Software Ver. 7. CA, USA). Negative controls included the omission of primary antibody and replacement with non-immune serum from secondary antibody host and inclusion of secondary antibodies.
Quantitative analysis of melanosome transfer: Fully-matched epidermal melanocytes (p4) and epidermal keratinocytes (p2) from 4 normal individuals (i.e., F39; F67; F54; F52) were maintained in 8-well Lab-Tek® chamber slides at a total cell density of 2×104 cells/well and in a ratio of 1 melanocyte to 10 normal keratinocytes. The co-culture was maintained for further 24 h before washing 3-times with sterile PBS before incubation with starved medium alone, or L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml) for 12 and 72 h. Controls included IBMX (100 μM) and niacinamide (10 μM). Cells were then processed for immunofluorescence staining with gp100 to assess melanosome transfer to keratinocytes.
Evaluation of melanosome transfer was performed by counting fluorescent gp100-positive spots within recipient keratinocytes in 5 random microscopic fields per well at 100× magnification (oil-immersion) in 3 independent experiments. To avoid counting melanin granules that may still be associated with melanocytes, we only counted gp100-positive spots within keratinocytes that were not in direct contact with melanocytes.
Knockdown of Myo-X using siRNA: A synthetic siRNA targeting human Myo-X (5′-CAGCGGTATAAGAGAAATCAA-3′) (SEQ ID No 1) and a non-silencing control, consisting of siRNA that has no known homology with mammalian genes (5′-AATTCTCCGAACGTGTCACGT-3′) (SEQ ID No 2) was obtained from Qiagen, Valencia, Calif. A day before siRNA treatment, 5×105 epidermal melanocytes per well were plated onto six-well plates at 50-60% confluency and incubated at 37° C. for 12 h. Cells were then treated for 12 h with a final concentration of 25 nM siRNA by using HiPerFect Transfection Reagent (Qiagen, Valencia, Calif.) following the manufacturer's instructions. Fluorescence microscopy was used to verify that approximately 100% of the cells (using non-silencing control labelled with alexa fluor 488 labels) had taken up the siRNA. At 12 h, the cells from each well were replated into 8-well Lab-Tek® chamber slides. For co-culture studies siRNA treated melanocytes were seeded with normal keratinocytes (passage 2) in 8-well Lab-Tek® chamber slides at a cell density of 2×104 cells/well and in a ratio of 1 siRNA treated melanocyte to 10 normal keratinocytes. At 24 h the co-culture were processed for double Immunofluorescence staining with gp100 and cytokeratin or Myo-X to study of the influence of gene knockdown on melanosome transfer, and parallel samples were assayed by immunofluorescence staining and western blot analysis to verify gene knockdown. For another experiment these co-culture were treated with or without L-18-23 for 24 h.
Results/Discussion
Dose Selection of Plant Extracts L-18-23:
Cells were incubated with the plant extracts and modulators of pigmentation (IBMX and niacinamide) at a range of concentrations. Some doses resulted in cell death or abnormal change in cell morphology (e.g., vacuolation). The inventor showed that optimal L-18-23 active doses that were not associated with altered cell growth or morphology. These doses were therefore used for the remainder of the study.
Normal human epidermal keratinocyte culture (Female-67; p2) were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and LVMH-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM) and niacinamide (10 μM).
Normal human epidermal melanocyte culture (Female-67; p4) were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM) and niacinamide (10 μM).
Dose selection for experimental DesignFinal dose selection for study on MC-Plant ExtractsKC Co-cultureL-180.001% L-191.0%L-200.5%L-21  10 μg/mlL-221.0%L-230.05 μg/mlEffect of Plant Extracts L-18-23 on Melanogenesis:
Normal human epidermal melanocytes (F52; p5) and human melanoma (FM55) were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), LVMH-22 (1%) and L-23 (0.05 μg/ml). In addition IBMX and NH4Cl (positive melanocytic modulators) and niacinamide (negative modulator) were used as controls. Visible change was not particularly evident in normal melanocyte due to their already high basal melanin levels (FIG. 1). By contrast, visible change was evident in melanoma cells basal melanin levels were low (FIG. 2). L-19, L-20, L-21 and L-23 significantly reduced melanin content compared with basal levels in both normal melanocytes and FM55 melanoma cells. L-22 however was associated with an increase in melanin content compared with basal levels. L-18 did not induce a significant change in these cells. Results are summarized in Table 1 and in FIGS. 1 & 2.
FIG. 1: Effect of Plant Extracts L-18-23 Actives on Melanogenesis in Normal Pigmented Human Melanocytes
Normal human epidermal melanocytes (Female-52; p5) were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM), NH4Cl (100 μM), and niacinamide (10 μM).
(A) Melanin content was determined spectro-photometrically (475 nm) after sodium hydroxide solubilise
(B) Visible change was not particularly evident in these cells due to their already high basal melanin levels. Results were expressed as change in melanin content (pg/cell) compared to unstimulated control levels. Means are ±SEM of 3 independent experiments with *P<0.05, **P<0.01, ***P<0.001. NS—Not Significant
FIG. 2: Effect of Plant Extracts L-18-23 on Melanogenesis in Moderately-Pigmented FM55 Melanoma
FM55 cells were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), LVMH-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM), NH4Cl (100 μM), and niacinamide (10 μM).
(A) Melanin content was determined spectrophotometrically (475 nm) after sodium hydroxide solubilisation. Cells with low basal melanin levels showed visible increases in melanogenesis after IBMX and NH4Cl stimulation.
(B). Results were expressed as the change in melanin content (pg/cell) compared to unstimulated control levels. Means are ±SEM of 3 independent experiments with *P<0.05, **P<0.01, ***P<0.001. NS—Not Significant.
TABLE 1Changes in melanin levels after incubation with plant extracts L-18-23BasalNCIBMXNH4ClL-18L-19L-20L-21L-22L-23F52 EM0−4.01 +74.57+166.17−4.57−34.43−15.66−43.68+31.60−25.25% change(35.63)(34.2)(62.2)(94.83)(34.04)(23.36)(30.62)(20.2)(46.89)(26.62)(pg/cell)FM550−5.94+250.8 +478.82−3.22−21.90−20.36−58.66+29.33−33.20% change(9.92)(9.33)(34.8)(56.83)(9.62)(7.74)(7.92)(4.2)(12.82)(6.63)(pg/cell)
Effect of Plants Extracts L-18-23 on Tyrosinase Activity:
Normal human epidermal melanocytes (F52; p5) and human melanoma (FM55) were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). In addition IBMX and NH4Cl (positive melanocytic modulators) and niacinamide (negative modulator) were used as controls. IBMX and NH4Cl were associated with a dramatic increase in dopa oxidase activity of tyrosinase in both EM and FM55 cells (FIGS. 3 & 4). Of the L-18-23, L-19, L-20, L-21 and L-23 significantly reduced tyrosinase activity in both normal melanocytes and FM55 melanoma cells compared with basal levels (FIGS. 3 & 4). By contrast, L-22 however was associated with a significant increase in tyrosinase activity compared with basal levels, while L-18 did not induce any significant change. These results are summarized in Table 2.
FIG. 3: Effect of Plant Extracts L-18-23 on Tyrosinase Activity in Pigmented Normal Human Melanocytes
Normal human epidermal melanocytes (Female-52; p5) were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), LVMH-22 (1%) and L-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM), NH4Cl (100 μM), and niacinamide (10 μM).
(A) Protein extracts were electroblotted and membranes stained with L-DOPA for the estimation of tyrosinase activity.
(B) Densitometric scanning of band intensities and values were expressed as a fold increase compared to unstimulated control levels Means are ±SEM of 3 independent experiments with *P<0.05, **P<0.01, ***P<0.001. NS—Not Significant.
FIG. 4: Effect of Plant Extracts L-18-23 on Tyrosinase Activity in Moderately Pigmented FM55 Melanoma.
FM55 cells were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM), NH4Cl (100 μM), and niacinamide (10 μM.).
(A) Protein extracts were electroblotted and membranes stained with L-DOPA for the estimation of tyrosinase activity.
(B) Densitometric scanning of band intensities and values were expressed as the fold increase compared to unstimulated control levels. Means are ±SEM of 3 independent experiments with *P<0.05, **P<0.01, ***P<0.001. NS—Not Significant.
TABLE 2Changes in tyrosinase activity after incubation with plants extracts L-18-23BasalNCIBMXNH4ClL-18L-19L-20L-21L-22L-23F52 EMc % 0−4.58+72.47+159.63−6.17−55.23−13.76−60.55+16.50−39.44changeFM55 % 0−3.31+141.72+408.27−5.29−50.37−22.15−42.54+43.54−42.88change
However, results from EM as monoculture may not fully reflect paracrine influences from keratinocyte and thus may not fully reveal L-18-23 associated effects. Therefore, the above assessment for tyrosinase activity was repeated with two of the plant extracts; one which showed no change (L-18) and one with a significant decrease (L-23) in fully-matched EM:KC co-cultures. It was interesting to note that under co-culture conditions, L-18 now showed a significant decrease in dopa oxidase activity of tyrosinase (from −3.46% when on EM cells grown alone to −57.02% when co-cultured with matched KC). For L-23, a further decrease in dopa oxidase activity of tyrosinase was seen when in co-culture (i.e., from −41.16% in monoculture to −76.17% in co-culture) (FIG. 5, and Table 3). A similar positive and negative trend was also seen with IBMX and niacinamide respectively.
FIG. 5: Effect of Plant Extracts L-18 and L-23, IBMX and Niacinamide on Tyrosinase Activity in Normal Human Melanocyte and Keratinocyte Co-Culture (Note: Protein Loading was Normalized for Melanocyte Protein Only).
Normal human epidermal melanocyte culture (Female-52; p5) and matched co-culture F52 MC-KC were treated for 72 h with L-18 (0.001%), L-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM), and niacinamide (10 μM). Negative control lane: Keratinocytes only
(A) Protein extracts were electroblotted and membranes stained with L-DOPA for the estimation of tyrosinase activity.
(B) Densitometric scanning of band intensities and values were expressed as the fold increase compared to unstimulated control levels. Means are ±SEM of 3 independent experiments with *P<0.05, **P<0.01, ***P<0.001.
TABLE 3Changes in EM tyrosinase activity on EM:KC co-cultures after incubationwith plants extracts L-18 and L-23BasalNCL-18L-23IBMXEMEM-EKEMEM-EKEMEM-EKEMEM-EKEMEM-EK% Change in EM0+12.16−2.12−20.50−3.46−57.02−41.16−76.17+56.51+93.41Tyrosinase Activityvs. Basal EM level(i.e. 0)% Change in EM+12.16−18.84−56.47−57+23.57Tyrosinase Activity of stimulated EM vs. stimulated EM-EK)level (i.e. 12.16)Quantitative Analysis of gp100-Positive Melanosome Transfer using Plants Extracts L-18 and L-23:
Gp100 immunostaining provides a powerful tracking method for the global assessment of melanin transfer to keratinocytes, and for the evaluation of melanocyte phenotype modulators (Singh et al., 2008). Double immunolabelling (NKi/beteb and anti-cytoskeleton antibody) of the EM:KC co-cultures after treatment with all L-18-23, a phosphodiesterase inhibitor IBMX (i.e., cAMP inducer) and niacinamide all revealed clear changes in the number of fluorescent green gp100-positive melanin granules in keratinocytes (FIG. 6). In this assay melanosome transfer from melanocytes to matched keratinocytes under basal (i.e., unstimulated) conditions was determined at an average of 27.3 gp100-positive spots per keratinocyte. However, this was increased almost 4-fold after 24 h stimulation of co-cultures with IBMX to 99.5 gp100-positive spots per keratinocyte. Conversely, niacinamide reduced melanosome transfer by 28% (19.6 gp100-positive spots per keratinocyte; p<0.01; a result that correlates with its clinical use as a lightener of cutaneous pigmentation (Hakozaki et al, 2002; Greatens et al., 2005).
L-18, L-19, L-20, L-21 and L-23 all significantly reduced melanosome transfer as evidenced by a reduction of melanosome transfer compare to unstimulated control. By contrast, an increase in melanosome transfer to keratinocytes was observed when co-cultures were stimulated with L-23), compared with unstimulated basal levels.
FIG. 6: Quantitative Analysis of Melanosome Transfer After Incubation with Plants Extracts L-18-23 (Table 4)
Melanocyte-keratinocyte matched co-cultures (Female-39) were treated for 24 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml) along with parallel known melanocytic modulators (IBMX (100 μM) and niacinamide (10 μM). Double-immunolabelling with anti-gp100 antibody (green) and anti-actin antibody (red) revealed clear changes in the number of fluorescent spots transferred to keratinocytes. Quantification of transferred melanosomes taken from 5 randomly-selected microscopic fields (total 20 cells per field) for each of the different treatment groups. Values were expressed as the percentage increase in the number of gp100-positive spots per keratinocyte compared to unstimulated control levels. Means are ±SEM of 4 independent experiments with **P<0.01, ***P<0.001.
TABLE 4gp 100+27.399.519.617.816.521.717.636.014.5Spots/KC±0.9±5.4±0.7±1.5±1.9±0.8±0.9±2.9±2.2% Change+264−28−34−39−20−35+31−47of MelaninTransfer vs.BasalEffect of Plant Extracts L-18-23 on Myo-X Expression in Normal Human Melanocytes.
Double immunolabeling with antibodies against the Myo-X (Date not shown) and gp100 revealed prominent Myo-X expression in EM cell periphery and dendritic tips. Myo-X was also detected in EM filopodia. We have found that IBMX increased the expression of Myo-X and the overall number of filopodia (as suggested by total number of yellow spots beyond EM dendritic tips). IBMX also enhanced Myo-X localization in EM filopodia compared with untreated cells in. By contrast, Myo-X expression was significantly down-regulated in EM by L-18, L-19, L-20, L-21 and L-23, while it was upregulated by L-22 alone. The localization of Myo-X within EM filopodia was also significantly reduced by these plant extracts.
Western blot analysis of EM (F39) stimulated for 12 h by IBMX (100 μM) showed a 3-fold increase in Myo-10 expression as compared with the untreated controls (FIG. 7A,B). By contrast, the melanosome transfer inhibitor niacinamide (10 μM) reduced Myo-X expression by 60%. All L-18-23 significantly down-regulated EM Myo-X expression (FIG. 7A,B), with the exception of L-22.
FIG. 7: Effect of Plant Extracts L-18-23 on Myo-X Protein Expression by Western Blotting in Normal Human Melanocytes
Normal human epidermal melanocyte cultures (F39-EM) were treated for 12 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). Controls: IBMX (100 μM) and niacinamide (10 μM).
(A) Cell extracts were analyzed by Western blotting using anti-Myo-X and anti-β-actin as a loading control.
(B) Densitometric analysis of Myo-X and actin band intensities expressed as percentage change compared to unstimulated controls. Means are ±SEM of 3 independent experiments with **P<0.01, ***P<0.001.
The Knockdown of Myo-X Expression in Epidermal Melanocytes Inhibits Melanosome Transfer to Epidermal Keratinocytes
The role of Myo-X in melanosome transfer was investigated by using synthetic siRNA for 12 h against human Myo-X and non-silencing control siRNA. Myo-X silencing in melanocyte cells was assayed by western blot analysis. This inhibition was also confirmed by immunofluorescence, which also showed that the expression of Myo-X was almost completely inhibited compared to the non-silencing control siRNA. Double-immunolabelling of the Myo-X-silenced melanocytes with anti-gp100 antibody revealed the absence of filopodia as evidenced by the lack of gp100-positive melanosome around the melanocyte tips. Melanocyte treated with non-silencing control siRNA showed prominent Myo-X and gp100 in the filopodial region.
We were keen to determine, whether endogenous Myo-X expression is a requirement for melanosome transfer via filopodia in melanocyte. In order to test the involvement of Myo-X in melanin transfer, Myo-X-silenced melanocytes and non-silenced melanocytes were used to establish the MC-KC co-culture for 24 h. Double Immunolabelling of this co-culture with anti-gp100 antibody and anti-Myo-X clearly showed the inhibition in the number of fluorescent spots transferred to keratinocytes cultured with Myo-X-silenced melanocytes. Double-immunolabelling with anti-gp100 antibody and anti-cyokeratin revealed clear changes in the number of fluorescent spots (representing melanin granule) transferred to keratinocytes.
Using this assay the rate of melanosome transfer from melanocytes to keratinocytes with non-silencing control siRNA conditions was determined (FIG. 8). This level of melanosome transfer was reduced by 80% in Myo-X-silenced melanocytes cultured with normal keratinocyte cells for 24 h (FIG. 8). This is the first demonstration of a physiological role of Myo-X in the successful transfer of melanosomes from melanocytes to keratinocytes.
In the following examples, the percentages are given by weight from the total weight of the composition. The amount of plant extracts is expressed as dry weight.